FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

Liver Transplantation Outcomes From Controlled Circulatory Death Donors: SCS vs in situ NRP vs ex situ NMP.

OBJECTIVE: To compare the outcomes of livers donated after circulatory death (DCD) and undergoing either in situ normothermic regional perfusion (NRP) or ex situ normothermic machine perfusion (NMP) with livers undergoing static cold storage (SCS). SUMMARY OF BACKGROUND DATA: DCD livers are associated with increased risk of primary nonfunction, poor function, and nonanastomotic strictures (NAS), leading to underutilization. METHODS: A single center, retrospective analysis of prospectively collected data on 233 DCD liver transplants performed using SCS, NRP, or NMP between January 2013 and October 2020. RESULTS: Ninety-seven SCS, 69 NRP, and 67 NMP DCD liver transplants were performed, with 6-month and 3-year transplant survival (graft survival non-censored for death) rates of 87%, 94%, 90%, and 76%, 90%, and 76%, respectively. NRP livers had a lower 6-month risk-adjusted Cox proportional hazard for transplant failure compared to SCS (hazard ratio 0.30, 95% Confidence Interval 0.08-1.05, P = 0.06). NRP and NMP livers had a risk-adjusted estimated reduction in the mean model for early allograft function score of 1.52 (P < 0.0001) and 1.19 (P < 0.001) respectively compared to SCS. Acute kidney injury was more common with SCS (55% vs 39% NRP vs 40% NMP; P = 0.08), with a lower risk-adjusted peak-to-baseline creatinine ratio in the NRP (P = 0.02). No NRP liver had clinically significant NAS in contrast to SCS (14%) and NMP (11%, P = 0.009), with lower risk-adjusted odds of overall NAS development compared to SCS (odds ratio = 0.2, 95%CI 0.06-0.72, P = 0.01). CONCLUSION: NRP and NMP were associated with better early liver function compared to SCS, whereas NRP was associated with superior preservation of the biliary system.

Bile Biochemistry Following Liver Reperfusion in the Recipient and Its Association With Cholangiopathy.

Cholangiocytes secrete bicarbonate and absorb glucose, producing bile with alkaline pH and low glucose content. These functions of cholangiocytes have been suggested as a marker of bile duct viability during normothermic ex situ liver perfusion, and they are now monitored routinely after reperfusion in our center. In this study, we reviewed the composition of bile immediately after reperfusion in liver transplant recipients to determine normal posttransplant parameters and the predictive value of bile biochemistry for the later development of cholangiopathy. After reperfusion of the liver graft, a cannula was placed in the bile duct to collect bile over a median 44-minute time period. The bile produced was analyzed using a point-of-care blood gas analyzer (Cobas b221, Roche Diagnostics, Indianapolis, IN). A total of 100 liver transplants (35 from donation after circulatory death and 65 from donation after brain death) were studied. Median bile pH was 7.82 (interquartile range [IQR], 7.67-7.98); median bile glucose was 2.1 (1.4-3.7) mmol/L; median blood-bile-blood pH difference was 0.50 (0.37-0.62); and median blood-bile glucose difference was 7.1 (5.6-9.1) mmol/L. There were 12 recipients who developed cholangiopathy over a median follow-up of 15 months (IQR, 11-20 months). Bile sodium (142 versus 147 mmol/L; P = 0.02) and blood-bile glucose concentration differences (5.2 versus 7.6 mmol/L; P = 0.001) were significantly lower and were associated with ischemic cholangiopathy. In conclusion, bile biochemistry may provide useful insights into cholangiocyte function and, hence, bile duct viability. Our results suggest bile glucose is the most sensitive predictor of cholangiopathy.

Infection and Prophylaxis During Normothermic Liver Perfusion: Audit of Incidence and Pharmacokinetics of Antimicrobial Therapy.

BACKGROUND: Ex situ normothermic liver perfusion (NMP) in a blood-based perfusate is associated with a risk of microbe growth, resulting in life-threatening posttransplant sepsis. Antibiotics are widely used, but the pharmacokinetics of these agents are unknown as is their efficacy. We wished to assess the perfusate concentrations of the meropenem and fluconazole that we use and to audit the incidence of infection with this antimicrobial therapy. METHODS: Fluconazole and meropenem (100 mg each) were added to the perfusate before NMP began, and serial samples were taken and assayed for drug concentrations. Perfusate cultures were available from 210 of the 242 perfusions performed between February 1, 2018, and April 6, 2023; these were reviewed. RESULTS: Following administration of 100 mg fluconazole, levels fell slightly from a median of 24.9 mg/L at 1 h to 22.6 mg/L at 10 h. In contrast, meropenem concentrations fell over time, from a median of 21.8 mg/L at 1 h to 9.4 mg/L at 10 h. There were 4 significant microorganisms grown in the perfusions, including 3 Candida species and an Enterococcus faecium . All the Candida -infected livers were transplanted with no adverse consequences, the recipients being treated with anidulafungin upon identification of the infecting organism; the Enterococcus -infected liver was not transplanted. CONCLUSIONS: Serious infection is a risk with NMP but appears to be mitigated with a protocol combining fluconazole and meropenem. This combination may not be appropriate in areas where resistance is prevalent. Routine culture of NMP perfusate is essential to identify breakthrough organisms early and enable recipient treatment.

Bile Chemistry During Ex Situ Normothermic Liver Perfusion Does Not Always Predict Cholangiopathy.

BACKGROUND: Bile chemistry during normothermic ex situ liver perfusion (NESLiP) has been suggested to be an indicator of cholangiopathy. The normal range of biochemical variables in bile of livers undergoing NESLiP has not been defined, nor have published biliary viability criteria been assessed against instances of posttransplant nonanastomotic bile strictures (NASs). METHODS: The bile and perfusate chemistry of 200 livers undergoing NESLiP between February 1, 2018, and October 30, 2023, was compared. In addition, 11 livers that underwent NESLiP and later developed NAS were selected and their bile chemistry was also examined. RESULTS: In livers that did not develop cholangiopathy, concentrations of sodium, potassium, and chloride were slightly higher in bile than in perfusate, whereas the concentration of calcium was slightly lower. Bile was alkali and had a lower glucose concentration than perfusate. Cholangiocyte glucose reabsorption was shown to saturate at high perfusate concentrations and was more impaired in livers donated after circulatory death than in livers donated after brain death. Published criteria failed to identify all livers that went on to develop NASs. CONCLUSIONS: A significant false-negative rate exists with current biliary viability criteria, probably reflecting the patchy and incomplete nature of the development of NASs in the biliary tree. The data presented here provide a benchmark for future assessment of bile duct chemistry during NESLiP.

Localized Liver Injury During Normothermic Ex Situ Liver Perfusion Has No Impact on Short-term Liver Transplant Outcomes.

BACKGROUND: Normothermic ex situ liver perfusion (NESLiP) has the potential to increase organ utilization. Radiological evidence of localized liver injury due to compression at the time of NESLiP, termed cradle compression, is a recognized phenomenon but is poorly characterized. METHODS: A retrospective analysis of a prospectively collected database was performed of transplanted livers that underwent NESLiP and subsequently had a computed tomography performed within the first 14 d posttransplant. The primary study outcome was 1-y graft survival. RESULTS: Seventy livers (63%) were included in the analysis. Radiological evidence of cradle compression was observed in 21 of 70 (30%). There was no difference in rate of cradle compression between donor after circulatory death and donated after brain death donors ( P  = 0.37) or with duration of NESLiP. Univariate analysis demonstrated younger (area under the receiver operating characteristic, 0.68; P = 0.008; 95% confidence interval [CI], 0.55-0.82) and heavier (area under the receiver operating characteristic, 0.80; P  < 0.001; 95% CI, 0.69-0.91) livers to be at risk of cradle compression. Only liver weight was associated with cradle compression on multivariate analysis (odds ratio, 1.003; P  = 0.005; 95% CI, 1.001-1.005). There was no difference in 1-y graft survival (16/17 [94.1%] versus 44/48 [91.6%]; odds ratio, 0.69; P  = 0.75; 95% CI, 0.07-6.62). CONCLUSIONS: This is the first study assessing the impact of cradle compression on outcome. We have identified increased donor liver weight and younger age as risk factors for the development of this phenomenon. Increasing utilization of NESLiP will result in the increased incidence of cradle compression but the apparent absence of long-term sequelae is reassuring. Routine postoperative axial imaging may be warranted.

D-dimer Release From Livers During Ex Situ Normothermic Perfusion and After In Situ Normothermic Regional Perfusion: Evidence for Occult Fibrin Burden Associated With Adverse Transplant Outcomes and Cholangiopathy.

BACKGROUND: Deceased donor livers are prone to biliary complications, which may necessitate retransplantation, and we, and others, have suggested that these complications are because of peribiliary vascular fibrin microthrombi. We sought to determine the prevalence and consequence of occult fibrin within deceased donor livers undergoing normothermic ex situ perfusion (NESLiP) and evaluate a role for fibrinolysis. METHODS: D-dimer concentrations, products of fibrin degradation, were assayed in the perfusate of 163 livers taken after 2 h of NESLiP, including 91 that were transplanted. These were related to posttransplant outcomes. Five different fibrinolytic protocols during NESLiP using alteplase were evaluated, and the transplant outcomes of these alteplase-treated livers were reviewed. RESULTS: Perfusate D-dimer concentrations were lowest in livers recovered using in situ normothermic regional perfusion and highest in alteplase-treated livers. D-dimer release from donation after brain death livers was significantly correlated with the duration of cold ischemia. In non-alteplase-treated livers, Cox proportional hazards regression analysis showed that D-dimer levels were associated with transplant survival ( P = 0.005). Treatment with alteplase and fresh frozen plasma during NESLiP was associated with significantly more D-dimer release into the perfusate and was not associated with excess bleeding postimplantation; 8 of the 9 treated livers were free of cholangiopathy, whereas the ninth had a proximal duct stricture. CONCLUSIONS: Fibrin is present in many livers during cold storage and is associated with poor posttransplant outcomes. The amount of D-dimer released after fibrinolytic treatment indicates a significant occult fibrin burden and suggests that fibrinolytic therapy during NESLiP may be a promising therapeutic intervention.

Predicting Early Allograft Function After Normothermic Machine Perfusion.

BACKGROUND: Normothermic ex situ liver perfusion is increasingly used to assess donor livers, but there remains a paucity of evidence regarding criteria upon which to base a viability assessment or criteria predicting early allograft function. METHODS: Perfusate variables from livers undergoing normothermic ex situ liver perfusion were analyzed to see which best predicted the Model for Early Allograft Function score. RESULTS: One hundred fifty-four of 203 perfused livers were transplanted following our previously defined criteria. These comprised 84/123 donation after circulatory death livers and 70/80 donation after brain death livers. Multivariable analysis suggested that 2-h alanine transaminase, 2-h lactate, 11 to 29 mmol supplementary bicarbonate in the first 4 h, and peak bile pH were associated with early allograft function as defined by the Model for Early Allograft Function score. Nonanastomotic biliary strictures occurred in 11% of transplants, predominantly affected first- and second-order ducts, despite selection based on bile glucose and pH. CONCLUSIONS: This work confirms the importance of perfusate alanine transaminase and lactate at 2-h, as well as the amount of supplementary bicarbonate required to keep the perfusate pH > 7.2, in the assessment of livers undergoing perfusion. It cautions against the use of lactate as a sole indicator of viability and also suggests a role for cholangiocyte function markers in predicting early allograft function.

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.